The present invention relates to a method for separating reversibly thermally precipitatable oligomeric N-substituted (meth)acrylamides and their conjugates from aqueous solution, as well as to separated, thermally precipitatable oligomeric N-substituted (meth)acrylamides and their conjugates, synthesized by the method.
In the area of polymer synthesis, radical telomerizing by means of a chain transfer reagent is a customary method of synthesizing a linear, low molecular weight polymer (having a degree of polymerization of less than 100 and molecular weights distributed very homogeneously) of controlled chain length and with a terminal functional group. By means of the functional group covalently bound conjugates, such as enzyme conjugates and affinity macroligands (AML), can be synthesized from the oligomers so produced.
A plurality of N-substituted (meth)acrylamides form water-soluble polymeric compounds, which precipitate reversibly from water above a lower critical demixing temperature, called the LCST (lower critical solution temperature). A list of monomers, which come into consideration, is given in the U.S. Pat. No. 5,162,582. The LCST is fixed by varying the monomer chemistry or the copolymer composition. It is, for example, 32° to 34° C. for poly-N-isopropylacrylamide in water. The LCST is independent of the chain length of the polymers and of the pH of the solution; as a rule, it is lowered by salts as a function of their molarity.
In methods known from the art, homogeneous, reversibly thermally precipitatable oligomers with terminal, functional groups are synthesized by telomerization in organic solvents or water. The yield is better than 60%. These oligomeric compounds are purified by repeated soluble-insoluble precipitations in organic solvents, such as acetone in hexane, with subsequent filtration and vacuum drying. The repeated purification is necessary in order to free the oligomeric preparation from the toxic monomers.
However, this purification method requires relatively large volumes of organic solvents having low water content. Experience has shown that, for a working-up step, approximately one liter of n-hexane with a water content of less than 0.05 percent is required in order to precipitate 10 g of oligomeric compound. The low water content of the organic solvent is required in order to avoid gelatinization of the oligomers during the precipitation and, with that, keep the oligomeric aggregates in a filtratable form. Accordingly, aside from the high costs for solvents and the use of many personnel for this purification method, which can be automated only with difficulty, this method is also very disadvantageous for reasons of operational safety and environmental protection, when used on an industrial scale with the volumes of solvents required for such a purpose.
The reversibly thermally precipitatable oligomers, the so-called “smart polymers” have a diversified area of applications in biotechnology, for example, in biocatalysis and bioseparation (affinity precipitation). For example, J. -P. Chen (J. Chem. Technol. Biotechnol. 73 (1998) 137–143) describes a conjugate with α-chymotrypsin, the enzyme activity and thermal stability of which are greater than those of native enzyme. For biocatalysis, such enzyme conjugates offer the advantages that the catalysis precedes homogeneously and that the biocatalysts can easily be separated by thermal precipitation and used once again.
Affinity precipitation is a bioseparation method, which utilizes the precipitation properties of the oligomers in combination with ligands, which have a specific affinity to a target substance, in order to separate and purify this target substance specifically.
In the WO 01/25287 A1, AML are described, which can be used efficiently for the purification of proteins and nucleic acids. However, the oligomers were produced only in small amounts and where purified either by precipitation in organic solvents or by diafiltration followed lyophilization. The latter method, however, is very time-consuming and can be used only for relatively small amounts. For the separation of thermally precipitatable oligomers and AML (including AML target substance complexes), centrifugation was selected because only small volumes were biopurified.
However, the industrial application of “smart polymers” in biocatalysis and bioseparation requires, on the one hand, a method for the synthesis of monomer-free oligomers on a large scale and, on the other, a method for the efficient separation of the thermal precipitates from a large volume. However, it is a common feature of all previously described examples of the separation of reversibly thermally precipitatable oligomers and their conjugates (enzyme conjugates, AML and AML target substances) from aqueous solution, that the precipitate separation was carried out by centrifugation using only using small volumes.
Y. G. Takei et al. (Bioconjugate Chem. 4 (1993) 42–46) have shown that, especially in the case of dilute solutions, low molecular weight oligomers require very high centrifugal accelerations, in order to separate more than 80 percent of the thermal precipitate. In the case of a 1 percent by weight solution of an oligomer with an average molecular weight of about 2,500 g/mole, only about 60 percent can be separated as precipitate even an a centrifugal acceleration of 10,000 g. Moreover, centrifugation has the disadvantage that it produces very compact precipitate gels, which can be dissolved again only very slowly. Moreover, it can be used only in batch operation and, with that, for relatively small volumes, since the thermal precipitate, because of its gelatinous consistency, cannot be supplied continuously.
Y. G. Takei et al. (Bioconjugate Chem. 4 (1993 42–46) have also shown that, in the case of oligomers with an average molecular weight of less than 5,000 g/mole, the filtration of the precipitate causes high losses. For example, only about 20 percent of precipitated oligomers with an average molecular weight of about 2,500 g/mole can be separated by filtration from a 1 percent by weight solution; at 10 percent by weight, the recovery rate increases to about 70 percent.
Accordingly, neither methods, which enable reversibly thermally precipitatable oligomers to be purified in large amounts without using organic solvents, nor methods, with which reversibly thermally precipitatable oligomers and their conjugates can be separated efficiently from aqueous solutions of large volume, are known from the state of the art.